Thermostable ribonuclease H from Archaeoglobus profundus

ABSTRACT

A polypeptide having an RNaseH activity from  Archaeoglobus profundus  and being highly useful in gene engineering; a gene encoding this polypeptide; and a genetic engineering process for producing the polypeptide.

TECHNICAL FIELD

The present invention relates to a polypeptide, specifically apolypeptide having a ribonuclease H activity which is highly valuablefor genetic engineering. The present invention also relates to a genethat is useful for producing said polypeptide by genetic engineering.The present invention further relates to a method for producing saidpolypeptide by genetic engineering.

BACKGROUND ART

There are endo-type and exo-type ribonucleases (RNA-degrading enzymes).Their substrate specificities are diverse, and they are involved incomplicated physiological activities. Enzymes such as ribonuclease T₁,ribonuclease T₂, ribonuclease H, ribonuclease P, ribonuclease I,ribonuclease II, ribonuclease III, ribonuclease IV, ribonuclease L areknown to have ribonuclease activities.

Ribonuclease H (hereinafter also referred to as RNase H) was firstisolated from calf thymus by W. H. Stein and P. Hausen in 1969. RNase Hsare currently classified into cellular RNase Hs and viral RNase Hs. Thecellular RNase Hs are widely present in eukaryotes such as variousanimal cells and yeasts and prokaryotes such as Escherichia coli,whereas the viral RNase Hs are present in RNA tumor viruses. Severalkinds of RNase H activities are present in a cell. They require divalentmetal ions such as Mg²⁺ and Mn²⁺.

An RNase H from Escherichia coli is a hydrolase that consists of 155amino acids, has a molecular weight of about 17 kDa and has a substratespecificity of specifically cleaving only the RNA strand in a DNA-RNAhybrid in an endo-type manner. The resulting oligomer has a phosphategroup at the 5′ end and a hydroxyl group at the 3′ end.

RNase HI and RNase HII have been identified as RNase Hs from E. coli. Ithas been shown that RNase HI has the following physiological functionsin the replication of the Col E1 plasmid: 1) it degrades RNAs bound toportions other than the normal replication origin to ensure the normalreplication origin; and 2) it synthesizes an RNA primer specific for thenormal replication origin. On the other hand, the function of RNase HIIremains unknown.

RNase Hs have uses as exemplified below based on the substratespecificities, and attention is paid to RNase Hs as very valuableenzymes:

1) removal of template mRNA upon cDNA cloning;

2) removal of poly(A) region in mRNA; and

3) fragmentation of RNA.

It is considered that RNase H increasingly becomes important with thedevelopment of genetic engineering. However, the expression level ofthis enzyme in E. coli is quite low. Then, production of this enzymeusing recombinant DNA techniques has been attempted. RNase Hs producedusing recombinant DNA techniques are now supplied from BRL, AmershamPharmacia Biotech, Takara Bio and the like.

These commercially available recombinant RNase Hs are produced usingEscherichia coli as a host (Kanaya et al., The Journal of BiologicalChemistry, 264:11546-11549 (1989)). A method of producing an RNase Hfrom a thermophile, which is much more stable than RNase H from E. coli,using E. coli has been reported (Kanaya et al., Dai 2 Kai NipponTanpakukougakukai Nenkai Program/Abstract (1990) pp. 69; Japanese PatentNo. 2533671). However, the enzymatic activity of the RNase H from athermophile produced using E. coli was lower than that of RNase H fromE. coli.

As described above, only thermostable RNase Hs whose productivities andenzymatic activities are lower than those of RNase H from E. coli areavailable. Thus, development of a thermostable RNase H whoseproductivity and enzymatic activity are equivalent to or more than thoseof the RNase H from E. coli has been desired for expanding the uses ofRNase H.

Then, RNase Hs having varying thermostabilities have been cloned inorder to solve the above-mentioned problems. Examples thereof includeRNase Hs derived from Bacillus caldotenax, Pyrococcus furriosus,Thermotoga maritima, Archaeoglobus fulgidus, Thermococcus litoralis,Thermococcus celer and Pyrococcus horikoshii as described in WO02/22831.

Further examples include RNase Hs derived from Thermus thermophilus(Nucleic Acids Research, Vol. 19, No. 16, p 4443-4449 (1991); U.S. Pat.No. 5,268,289), Pyrococcus furiosus (U.S. Pat. No. 5,610,066),Pyrococcus sp. KOD1 (JP-A 11-32772) and Archaeoglobus fulgidus (Journalof Molecular Biology, Vol. 307, p 541-556 (2001)).

However, there have been many problems to be improved concerning theseRNase Hs such as low thermostability, decreased activity on a DNA-RNAhybrid having short RNA strand and low specific activity. Then,development of further thermostable RNase H having distinct substratespecificity or mode of action has been desired to meet the importance ofRNase H which is considered to be increased more and more with thedevelopment of genetic engineering.

DISCLOSURE OF INVENTION

The main object of the present invention is to provide a polypeptidehaving an RNase H activity which is highly valuable for geneticengineering, a gene encoding said polypeptide and a method for producingsaid polypeptide by genetic engineering.

In view of the circumstances as described above, the present inventorshave studied intensively and conducted screening in order to obtain athermostable RNase H. As a result, the present inventors have found athermostable RNase H polypeptide having a high RNase H activity.Furthermore, the present inventors have found that the productivity ofthe thus obtained thermostable RNase H in production using geneticengineering techniques is high. Thus, the present invention has beencompleted.

The present invention is outlines as follows. The first aspect of thepresent invention relates to a polypeptide having a thermostableribonuclease H activity, selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO:1;

(b) a polypeptide having an amino acid sequence in which at least oneamino acid residue is deleted, added, inserted or substituted in theamino acid sequence of SEQ ID NO:1; and

(c) a polypeptide having an amino acid sequence that shares at least 54%homology with the amino acid sequence of SEQ ID NO:1.

The second aspect of the present invention relates to a nucleic acidencoding a polypeptide having a thermostable ribonuclease H activity,selected from the group consisting of:

(a) a nucleic acid encoding a polypeptide having the amino acid sequenceof SEQ ID NO:1;

(b) a nucleic acid encoding a polypeptide having an amino acid sequencein which at least one amino acid residue is deleted, added, inserted orsubstituted in the amino acid sequence of SEQ ID NO:1;

(c) a nucleic acid encoding a polypeptide having an amino acid sequencethat shares at least 54% homology with the amino acid sequence of SEQ IDNO:1;

(d) a nucleic acid having the nucleotide sequence of SEQ ID NO:2;

(e) a nucleic acid consisting of a nucleotide sequence in which at leastone nucleotide is deleted, added, inserted or substituted in thenucleotide sequence of SEQ ID NO:2 such that the deletion, addition,insertion or substitution of the nucleotide results in translation intoan amino acid sequence;

(f) a nucleic acid capable of hybridizing to any one of the nucleicacids of (a) to (d) or complementary strands thereof under stringentconditions; and

(g) a nucleic acid having a nucleotide sequence that shares at least 61%homology with the nucleotide sequence of SEQ ID NO:2.

The third aspect of the present invention relates to a recombinant DNAcomprising the nucleic acid of the second aspect.

The fourth aspect of the present invention relates a transformanttransformed with the recombinant DNA of the third aspect.

The fifth aspect of the present invention relates to a method forproducing a polypeptide having a thermostable ribonuclease H activity,the method comprising:

culturing the transformant of the fourth aspect; and

collecting a polypeptide having a thermostable ribonuclease H activityfrom the culture.

The sixth aspect of the present invention relates a polypeptide having athermostable ribonuclease H activity, obtainable by culturing atransformant into which a plasmid pApr108 is transferred. An Escherichiacoli strain harboring this plasmid is deposited under Budapest Treaty onAug. 20, 2002 (date of original deposit) under accession number FERMBP-8433 at International Patent Organism Depositary, National Instituteof Advanced Industrial Science and Technology, AIST Tsukuba Central 6,1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-8566, Japan.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the thermostability of the RNase H of the presentinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

As used herein, an RNase H refers to a hydrolase that has a substratespecificity of specifically cleaving only the RNA strand in a DNA-RNAhybrid in an endo-type manner, wherein the oligomer resulting from thecleavage has a phosphate group at the 5′ end and a hydroxyl group at the3′ end.

Although it is not intended to limit the present invention, having athermostable RNase H activity as used herein with respect to apolypeptide means that the polypeptide has an RNase H activity afterincubating it at a temperature of 70° C. or above for 15 minutes.

For example, a thermostable RNase H activity can be determined asfollows.

1 mg of poly(rA) or poly(dT) (both from Amersham Pharmacia Biotech) isdissolved in 1 ml of 40 mM tris-HCl (pH 7.7) containing 1 mM EDTA toprepare a poly(rA) solution and a poly(dT) solution.

The poly(rA) solution (to a final concentration of 20 μg/ml) and thepoly(dT) solution (to a final concentration of 30 μg/ml) are then addedto 40 mM tris-HCl (pH 7.7) containing 4 mM MgCl₂, 1 mM DTT, 0.003% BSAand 4% glycerol. The mixture is reacted at 37° C. for 10 minutes andthen cooled to 4° C. at prepare a poly(rA)-poly(dT) solution.

1 μl of an enzyme solution is added to 100 μl of the poly(rA)-poly(dT)solution. The mixture is reacted at 40° C. for 10 minutes. 10 μl of 0.5M EDTA is added thereto to terminate the reaction. Absorbance at 260 nmis then measured. As a control, 10 μl of 0.5 M EDTA is added to thereaction mixture, the resulting mixture is reacted at 40° C. for 10minutes, and the absorbance is then measured. A value (difference inabsorbance) is determined by subtracting the absorbance for the controlfrom the absorbance for the reaction in the absence of EDTA. Thus, theconcentration of nucleotide released from poly(rA)-poly(dT) hybrid bythe enzymatic reaction is determined on the basis of the difference inabsorbance. Thus, the thermostable RNase H activity according to thepresent invention can be determined.

Alternatively, the thermostable RNase H activity according to thepresent invention can be determined as follows. 100 μl of a reactionmixture [20 mM HEPES-potassium hydroxide (pH 8.5), 0.01% bovine serumalbumin (Takara Bio), 1% dimethyl sulfoxide, 4 mM magnesium acetate, 20μg/ml poly(dT) (Amersham Pharmacia Biotech), 30 μg/ml poly(rA) (AmershamPharmacia Biotech)] which has been incubated at 40° C. is added to 1 μlof an enzyme solution whose activity is to be determined. The mixture isreacted at 40° C. for 10 minutes. The reaction is then terminated byadding 10 μl of 0.5 M EDTA (pH 8.0). Absorbance at 260 nm is thenmeasured.

One unit of RNase H is defined as an amount of enzyme that increasesA₂₆₀ corresponding to release of 1 nmol of ribonucleotide in 10 minuteswhich can be calculated according to the following equation:Unit=[Difference in Absorbance×Reaction Volume(ml)]/0.0152

The polypeptide of the present invention is exemplified by a polypeptidehaving the amino acid sequence of SEQ ID NO:1. The present inventionalso encompasses a polypeptide having an amino acid sequence in which atleast one amino acid residue is deleted, added, inserted or substitutedin the amino acid sequence of SEQ ID NO:1 as long as it exhibits athermostable RNase H activity.

A mutation such as deletion, insertion, addition or substitution of anamino acid in an amino acid sequence may be generated in a naturallyoccurring polypeptide. Such mutation may be generated due to apolymorphism or a mutation of the DNA encoding the polypeptide, or dueto a modification of the polypeptide in vivo or during purificationafter synthesis. However, it is known that such a mutated polypeptidemay exhibit a physiological or biological activity substantiallyequivalent to that of a polypeptide without a mutation if such amutation is present in a portion that is not important for the retentionof the activity or the structure of the polypeptide.

This is applicable to a polypeptide in which such a mutation isartificially introduced into an amino acid sequence of a polypeptide. Inthis case, it is possible to generate more various mutations. Forexample, it is known that a polypeptide in which a cysteine residue inthe amino acid sequence of human interleukin-2 (IL-2) is replaced by aserine retains the interleukin-2 activity (Science, 224:1431 (1984)).

Furthermore, it is known that certain polypeptides have peptide regionsthat are not indispensable to their activities. Such peptide regions areexemplified by a signal peptide in a polypeptide to be secretedextracellularly, or a prosequence or pre-prosequence found in aprecursor of a protease. Most of such regions are removed aftertranslation or upon conversion into an active polypeptide. Such apolypeptide has a primary structure different from that of a polypeptidewithout the region to be removed, but finally exhibits an equivalentfunction.

A gene having the nucleotide sequence of SEQ ID NO:2 which is isolatedaccording to the present invention encodes a polypeptide having theamino acid sequence of SEQ ID NO:1. This polypeptide has a thermostableRNase H activity. The polypeptide of the present invention includes apolypeptide from which a peptide region that is not indispensable to itsactivity has been deleted therefrom.

When a polypeptide is produced by genetic engineering, a peptide chainthat is irrelevant to the activity of the polypeptide of interest may beadded at the amino terminus or the carboxyl terminus of the polypeptide.For example, a fusion polypeptide, in which a portion of an aminoterminus region of a polypeptide that is expressed at a high level inthe host to be used is added at the amino terminus of the polypeptide ofinterest, may be prepared in order to increase the expression level ofthe polypeptide of interest. In another case, a peptide having anaffinity for a specific substance may be added at the amino terminus orthe carboxyl terminus of the polypeptide of interest in order tofacilitate the purification of the expressed polypeptide. The addedpeptide may remain added if it does not have a harmful influence on theactivity of the polypeptide of interest. If necessary, it may beengineered such that it can be removed from the polypeptide of interestby appropriate treatment, for example, by limited digestion with aprotease.

Thus, a polypeptide having an amino acid sequence in which at least oneamino acid residue is deleted, inserted, added or substituted in theamino acid sequence of SEQ ID NO:1 disclosed herein is encompassed bythe present invention if it has a thermostable RNase H activity.

Furthermore, a polypeptide having an amino acid sequence that shares atleast 54%, preferably 60%, more preferably 70%, most preferably 85%homology with the amino acid sequence of SEQ ID NO:1 disclosed herein isencompassed by the present invention if it has a thermostable RNase Hactivity.

The homology can be determined using, for example, a computer programDNASIS-Mac (Takara Bio), a computer algorithm FASTA (version 3.0;Pearson, W. R. et al., Pro. Natl. Acad. Sci., 85:2444-2448, 1988) or acomputer algorithm BLAST (version 2.0, Altschul et al., Nucleic AcidsRes. 25:3389-3402, 1997).

For example, a polypeptide having an amino acid sequence that shares atleast 54% homology with the amino acid sequence of the ribonuclease Hfrom Archaeoglobus profundus (SEQ ID NO:1) is encompassed by the presentinvention if it has a thermostable RNase H activity.

The polypeptide of the present invention can be produced, for example,by (1) purification from a culture of a microorganism producing thepolypeptide of the present invention, or (2) purification from a cultureof a transformant containing a nucleic acid encoding the polypeptide ofthe present invention.

(1) Purification from Culture of Microorganism Producing the Polypeptideof the Present Invention

The microorganism producing the polypeptide of the present invention isexemplified by Archaeoglobus profundus (DSM5631) which can be purchasedfrom Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. Themicroorganism is cultured under conditions suitable for the growth ofthe microorganism. Preferably, culture conditions that increase theexpression level of the polypeptide of interest are used. Thepolypeptide of interest produced in the cells or the culture medium canbe purified according to a method conventionally used for purifying aprotein.

A method conventionally used for culturing a thermostable bacterium canbe utilized for the cultivation of the above-mentioned strain. Nutrientsthat can be utilized by the strain are added to the culture medium. Forexample, starch can be used as a carbon source, and Tryptone, peptoneand yeast extract can be used as nitrogen sources. A metal salt such asa magnesium salt, a sodium salt or an iron salt may be added to aculture medium as a trace element. In addition, it may be advantageousto use artificial seawater for the preparation of a culture medium incase of a thermostable marine bacterium, for example.

The culture may be a standing culture or a spinner culture. For example,a dialysis culture method as described in Applied and EnvironmentalMicrobiology, 55:2086-2088 (1992) may be used. It is preferable todetermine the culture conditions and the cultivation time depending onthe strain or the composition of the culture medium to be used such thatthe productivity of the polypeptide becomes maximum.

A cell-free extract is first prepared in order to obtain a polypeptide.The cell-free extract can be prepared, for example, by collecting cellsfrom a culture by centrifugation, filtration or the like and thendisrupting the cells. A cell disruption method highly effective forextracting the enzyme of interest may be selected from sonication,disruption using beads, treatment with a lytic enzyme and the like. Ifthe polypeptide is secreted into a culture supernatant, the polypeptidein the culture supernatant is concentrated by ammonium sulfateprecipitation, ultrafiltration or the like. The concentrated polypeptideis used as a cell-free extract. A method conventionally used forpurifying a protein can be used to isolate the polypeptide from the thusobtained cell-free extract. For example, ammonium sulfate precipitation,ion exchange chromatography, hydrophobic chromatography, gel filtrationchromatography and the like can be used in combination.

(2) Purification from Culture of Transformant Transformed withRecombinant DNA Containing Nucleic Acid Encoding the Polypeptide of thePresent Invention

The polypeptide of the present invention can be obtained from atransformant transformed with a recombinant DNA that contains a nucleicacid encoding the polypeptide of the present invention, for example, anucleic acid having a nucleotide sequence of SEQ ID NO:2. A polypeptidehaving an amino acid sequence of SEQ ID NO:1 is produced using a nucleicacid having a nucleotide sequence of SEQ ID NO:2.

The polypeptide of the present invention may be purified from a cultureobtained by culturing a transformant into which the plasmid of thepresent invention, pApr108, is transferred.

There is no specific limitation concerning the host to be transformed.Examples thereof include those conventionally used in the field ofrecombinant DNA such as Escherichia coli, Bacillus subtilis, yeast,filamentous fungi, plants, animals, cultured plant cells and culturedanimal cells.

For example, the polypeptide of the present invention can be obtained byculturing Escherichia coli harboring a plasmid in which the nucleic acidof the present invention is linked downstream from a lac promoter or aT7 phage promoter under conventional culture conditions, for example, inLB medium (10 g/l Tryptone, 5 g/l yeast extract, 5 g/l NaCl, pH 7.2)containing 100 μg/ml of ampicillin at 37° C. until logarithmic growthphase, adding isopropyl-β-D-thiogalactopyranoside at a finalconcentration of 1 mM thereto and further culturing at 37° C. to expressthe polypeptide in the cultured cells.

Cells are collected by centrifugation after cultivation, disrupted bysonication, and a supernatant collected by centrifugation is used as acell-free extract. This cell-free extract exhibits a thermostable RNaseH activity. The polypeptide of the present invention can be purifiedfrom the cell-free extract by using known methods such as ion exchangechromatography, gel filtration, hydrophobic chromatography and ammoniumsulfate precipitation. Naturally, a partially purified product obtainedduring the purification process as described above also exhibits anRNase H activity. Since the polypeptide of the present inventionexpressed in Escherichia coli harboring a plasmid linked to the nucleicacid of the present invention is thermostable, it may be purified asfollows. For example, the cultured cell and/or the cell-free extract isheated at a temperature of 40° C. or above for about 10 minutes, andheat-denatured insoluble proteins derived from the host is removed. Anoptimal temperature or time may be suitably selected for the heattreatment.

As described above, when the polypeptide of the present invention isexpressed at normal temperature (e.g., 37° C.) using a transformantharboring a nucleic acid encoding the polypeptide, the resultingexpression product retains the activity, the thermostability and thelike. That is, the polypeptide of the present invention can assume itsinherent higher-order structure even if it is expressed at a temperaturequite different from the growth temperature of the original producercell.

The nucleic acid of the present invention is a nucleic acid that encodesthe polypeptide of the present invention. Specifically, it is (1) anucleic acid that encodes a polypeptide having the amino acid sequenceof SEQ ID NO:1, or an amino acid sequence in which at least one aminoacid residue is deleted, added, inserted or substituted in the sequenceand having a thermostable RNase H activity; (2) a nucleic acid havingthe nucleotide sequence of SEQ ID NO:2; (3) a nucleic acid that has anucleotide sequence that is capable of hybridizing to the nucleic acidof (1) or (2) above under stringent conditions, or that shares at least61%, preferably 70%, more preferably 80%, most preferably 90% homologywith the nucleotide sequence of (1) or (2) above, and that encodes apolypeptide having a thermostable RNase H activity, or the like.

The homology of the nucleotide sequence can be determined using acomputer program DNASIS-Mac, or a computer algorithm FASTA (version 3.0)or BLAST (version 2.0).

As used herein, a nucleic acid means a single-stranded ordouble-stranded DNA or RNA. If the nucleic acid of (2) above is an RNA,it is represented by a nucleotide sequence in which T is replaced by Uin the nucleotide sequence of SEQ ID NO:2, for example.

For example, the nucleic acid of the present invention can be obtainedas follows.

The nucleic acid of (2) above having the nucleotide sequence of SEQ IDNO:2 can be isolated as follows. A genomic DNA is prepared according toa conventional method from Archaeoglobus profundus (DSM5631) cultured asdescribed above for the polypeptide of the present invention. Thegenomic DNA is used to construct a DNA library. The nucleic acid can beisolated from the DNA library. Also, the nucleic acid can be obtained byamplifying a nucleic acid having a nucleotide sequence of SEQ ID NO:2 bya polymerase chain reaction (PCR) using the genomic DNA as a template.

Furthermore, a nucleic acid encoding a polypeptide having a thermostableRNase H activity similar to that of the polypeptide of the presentinvention can be obtained on the basis of the nucleotide sequence of thenucleic acid encoding the polypeptide of the present invention providedby the present invention (e.g., the nucleotide sequence of SEQ ID NO:2).Specifically, a DNA encoding a polypeptide having a thermostable RNase Hactivity can be screened, using the nucleic acid encoding thepolypeptide of the present invention or a portion of the nucleotidesequence as a probe for hybridization, from a DNA extracted from cells,PCR products obtained using the DNA as a template or the like.Alternatively, a DNA encoding a polypeptide having a thermostable RNaseH activity can be amplified using a gene amplification method such as aPCR using a primer designed based on the above-mentioned nucleotidesequence. Additionally, a DNA encoding a polypeptide having athermostable RNase H activity can be chemically synthesized. The nucleicacids of (1) or (3) above can be obtained according to such a method.

A nucleic acid fragment containing only a portion of the nucleic acid ofinterest may be obtained according to the above-mentioned method. Inthis case, the entire nucleic acid of interest can be obtained asfollows. The nucleotide sequence of the obtained nucleic acid fragmentis determined to confirm that the fragment is a portion of the nucleicacid of interest. Hybridization is carried out using the nucleic acidfragment or a portion thereof as a probe. Alternatively, a PCR iscarried out using a primer synthesized on the basis of the nucleotidesequence of the nucleic acid fragment.

“Hybridizing under stringent conditions” means that a nucleic acid iscapable of hybridizing under conditions as described in T. Maniatis etal. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., Cold SpringHarbor Laboratory (1989) or the like. For example, it refers tocapability of hybridization under the following conditions. A membraneonto which a nucleic acid is immobilized is incubated with a probe in6×SSC (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone,0.1% Ficoll 400 and 0.01% denatured salmon sperm nucleic acid at 50° C.for 12 to 20 hours. After incubation, the membrane is washed in 2×SSCcontaining 0.5% SDS at 37° C. while changing the SSC concentration downto 0.1× and the temperature up to 50° C. until the signal from theimmobilized nucleic acid can be distinguished from background, and theprobe is then detected. The activity of the protein encoded by the thusobtained novel nucleic acid is determined as described above, therebyconfirming whether or not the nucleic acid is the nucleic acid ofinterest.

If an oligonucleotide probe is to be used, “stringent conditions” referto, for example, incubation at a temperature of [Tm−25° C.] overnight ina solution containing 6×SSC, 0.5% SDS, 5×Denhardt's and 0.01% denaturedsalmon sperm nucleic acid although it is not intended to limit thepresent invention.

Tm of an oligonucleotide probe or primer can be determined, for example,according to the following equation:Tm=81.5−16.6(log₁₀ [Na⁺])+0.41(% G+C)−(600/N)wherein N is the chain length of the oligonucleotide probe or primer; %G+C is the content of guanine and cytosine residues in theoligonucleotide probe or primer.

If the chain length of the oligonucleotide probe or primer is shorterthan 18 bases, Tm can be estimated, for example, as the sum of theproduct of the number of A+T (adenine and thymine) residues multipliedby 2(° C.) and the product of the number of G+C residues multiplied by4(° C.):[(A+T)×2+(G+C)×4]

According to the present invention, a nucleic acid that is capable ofhybridizing to the nucleic acid encoding the polypeptide of the presentinvention under stringent conditions is encompassed by the presentinvention as long as it encodes a polypeptide having a thermostableRNase H activity even if it does not have the same nucleotide sequenceas that disclosed herein, as described above.

It is known that one to six codon(s) (a combination of three bases),which defines an amino acid in a gene, is assigned for each amino acid.Thus, many nucleic acids can encode one specific amino acid sequencealthough it depends on the amino acid sequence. Nucleic acids are notnecessarily stable in the nature. Generation of a mutation in anucleotide sequence is not unusual. A mutation generated in a nucleicacid may not alter the encoded amino acid sequence (called a silentmutation). In this case, it can be said that a different nucleic acidencoding the same amino acid sequence is generated. Thus, one cannotdeny the possibility that various nucleic acids encoding the same aminoacid sequence can be generated in the course of passage of an organismcontaining an isolated nucleic acid encoding one specific amino acidsequence. Furthermore, it is not difficult to artificially producevarious nucleic acids encoding the same amino acid sequence if one usesvarious genetic engineering techniques.

For example, if a codon used in an original nucleic acid encoding aprotein of interest is one whose codon usage is low in the host to beused for producing the protein by genetic engineering, the expressionlevel of the protein may be low. In this case, the codon is artificiallyconverted into one frequently used in the host without altering theencoded amino acid sequence aiming at elevating the expression level ofthe protein of interest (e.g., JP-B 7-102146). It is needless to saythat various nucleic acids encoding one specific amino acid sequence canbe artificially prepared as described above. They may also be generatedin the nature.

The nucleic acid encoding the polypeptide of the present invention(e.g., a nucleic acid having the nucleotide sequence of SEQ ID NO:2) canbe ligated to an appropriate vector to construct a recombinant DNA.There is no specific limitation concerning the vector to be used for theconstruction of the recombinant DNA. For example, plasmid vectors, phagevectors and virus vectors can be used. A suitable vector for the objectof the recombinant DNA is selected.

Furthermore, a transformant can be produced by transferring therecombinant DNA into an appropriate host. There is no specificlimitation concerning the host to be used for the production of atransformant. Microorganisms such as bacteria, yeasts and filamentousfungi as well as cultured cells from animals, plants, insects and thelike can be used. The polypeptide of the present invention can beproduced in large quantities by culturing the transformant to producethe polypeptide of the present invention in the culture.

(3) Polypeptide of the Present Invention

The polypeptide of the present invention obtained as described above canbe utilized for a nucleic acid amplification method, for example, asdescribed in WO 00/56877 or WO 02/16639. RNase H activities of some ofconventional RNase Hs may be reduced depending on the length of RNAportion in a chimeric nucleic acid (e.g., DNA-RNA-DNA). The polypeptideof the present invention is less susceptible to influence of the lengthof RNA portion. Thus, it can be preferably used for the basesubstitution detection method as described in WO 02/064833 or thecycling probe method as described in BioTechniques, Vol. 20, No. 2, p240-248 (1996). Since the polypeptide of the present invention has aproperty that its RNase H activity can be retained after treatment at95° C. for 15 minutes, it can be used for a variety of uses.

EXAMPLES

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Example 1 Cloning of Archaeoglobus profundus RNase H Gene

(1) Preparation of Genomic DNA from Archaeoglobus profundus

Cells of Archaeoglobus profundus (purchased from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH; DSM5631) collected from 10 ml ofa culture were suspended in 100 μl of a mixture containing 20% sucroseand 50 mM tris-HCl (pH 8.0). 20 μl of 0.5 M EDTA and 10 μl of 10 mg/mlaqueous solution of lysozyme chloride (Nacalai Tesque) were addedthereto. The mixture was reacted at 20° C. for 2 hours. After reaction,800 μl of a mixture containing 150 mM NaCl, 1 mM EDTA and 20 mM tris-HCl(pH 8.0), 10 μl of 20 mg/ml proteinase K (Takara Bio) and 50 μl of 10%aqueous solution of sodium lauryl sulfate were added to the reactionmixture. The mixture was incubated at 37° C. for 1 hour. After reaction,the mixture was subjected to phenol-chloroform extraction, ethanolprecipitation and air-drying. The precipitate was then dissolved in 50μl of TE to obtain a genomic DNA solution.

(2) Cloning of Middle Portion of RNase H Gene

Oligonucleotides RN-F1 (SEQ ID NO:3) and RN-R2 (SEQ ID NO:4) weresynthesized on the basis of portions conserved among amino acidsequences of various thermostable RNase Hs.

A PCR was carried out in a volume of 100 μl using 5 μl of theArchaeoglobus profundus genomic DNA solution prepared in Example 1-(1)as a template, and 100 μmol each of RN-F1 and RN-R2 as primers. TaKaRaEx Taq (Takara Bio) was used as a DNA polymerase for the PCR accordingto the attached protocol. The PCR was carried out as follows: 50 cyclesof 94° C. for 30 seconds, 45° C. for 30 seconds and 72° C. for 1 minute.After reaction, the reaction mixture was subjected to Microcon-100(Takara Bio) for removal of primers and concentration of the reactionmixture to obtain an about 0.5-kb DNA fragment AprF1R2.

(3) Cloning of Upstream and Downstream Portions of RNase H Gene

The nucleotide sequence of the about 0.5-kb fragment AprF1R2 obtained inExample 1-(2) was determined. A specific oligonucleotide AprRN-1 (SEQ IDNO:5) for cloning the upstream portion and a specific oligonucleotideAprRN-2 (SEQ ID NO:6) for cloning the downstream portion weresynthesized on the basis of the determined nucleotide sequence. Inaddition, 48 primers as shown in Table 1 were synthesized. The tagsequence in Table 1 is shown in SEQ ID NO:7.

TABLE 1 5′-tag sequence-NN-SSSSSSS-3′ (N: mixture of G, A, T and C; Srepresents the nucleotide sequence below) Nucleotide No. sequence  1ggagcag  2 ggcaaag  3 ggcaacg  4 ggcacag  5 ggcattg  6 ggccaag  7ggccttg  8 ggctaag  9 ggctacg 10 ggctcag 11 ggctttg 12 gggacag 13gggcaag 14 gggcttg 15 gggtacg 16 ggtaacg 17 ggtacgg 18 ggtagcg 19gtaacgg 20 gtaagcg 21 gtacacg 22 gtagacg 23 gtagcgg 24 gtcaacg 25gcaccag 26 gcagacg 27 gcagcag 28 gcatggg 29 gccaaag 30 gccacag 31gccattg 32 gcccaag 33 gcccttg 34 gcctacg 35 gcctcag 36 gcctttg 37gcgcaag 38 gcgcttg 39 gcggacg 40 gcgtaag 41 gctacgg 42 gctcacg 43gctccag 44 gcttgcg 45 gcttggg 46 ggacacg 47 ggaccag 48 ggagacg

PCRs were carried out in reaction mixtures containing 1 μl of theArchaeoglobus profundus genomic DNA solution prepared in Example 1-(1)as a template, a combination of 20 pmol of AprRN-1 or 20 pmol of AprRN-2and 20 pmol of one of the 48 primers listed in Table 1, 20 mMtris-acetate (pH 8.5), 50 mM potassium acetate, 3 mM magnesium acetate,0.01% BSA, 30 μM each of dNTPs and 2.5 units of TaKaRa Ex Taq DNApolymerase (Takara Bio). PCRs were carried out as follows: incubation at94° C. for 3 minutes; and 40 cycles of 98° C. for 10 seconds, 50° C. for10 seconds and 72° C. for 40 seconds. A portion of each PCR product wassubjected to agarose gel electrophoresis. Microcon-100 (Takara Bio) wasused to remove primers from reaction mixtures selected for thegeneration of single bands and to concentrate the reaction mixtures. Theconcentrates were subjected to direct sequencing to screen for fragmentscontaining the upstream or downstream portion of the RNase H. As aresult, it was shown that an about 600-bp PCR-amplified fragment Apr-1A5contained the upstream portion of the RNase H gene and an about 500-bpPCR-amplified fragment Apr-2D9 contained the downstream portion.

(4) Cloning of Entire RNase H Gene

Primers AprNde (SEQ ID NO:8) and AprBam (SEQ ID NO:9) were synthesizedon the basis of the nucleotide sequence.

A PCR was carried out in a volume of 100 μl using 1 μl of theArchaeoglobus profundus genomic DNA solution obtained in Example 1-(1)as a template, and 20 pmol each of AprNde and AprBam as primers. Ex TaqDNA polymerase (Takara Bio) was used as a DNA polymerase for the PCRaccording to the attached protocol. The PCR was carried out as follows:40 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for1 minute. An about 0.7-kb amplified DNA fragment was digested with NdeIand BamHI (both from Takara Bio). Then, plasmids pAPR111Nd and pApr108were constructed by incorporating the resulting DNA fragment betweenNdeI and BamHI sites in a plasmid vector pTV119Nd (a plasmid in whichthe NcoI site in pTV119N is converted into a NdeI site) or pET3a(Novagen), respectively.

(5) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase H Gene

The nucleotide sequences of the DNA fragments inserted into pAPR111Ndand pApr108 obtained in Example 1-(4) were determined according to adideoxy method.

Analyses of the determined nucleotide sequences revealed the existenceof an open reading frame presumed to encode RNase H. The nucleotidesequence of the open reading frame in pApr108 is shown in SEQ ID NO:2.The amino acid sequence of RNase H deduced from the nucleotide sequenceis shown in SEQ ID NO:1. The PCR fragments AprF1R2, Apr-1A5 and Apr-2D9correspond to a region from the 11th to 449th nucleotides in SEQ IDNO:2, a region from the 59th nucleotide toward the 5′ end, and a regionfrom the 373rd nucleotide toward the 3′ end, respectively. Escherichiacoli HMS174DE3 transformed with the plasmid pApr108 is designated andindicated as Escherichia coli HMS174/pApr108, and deposited on Aug. 20,2002 (date of original deposit) at International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki 305-8566, Japan under accession number FERM BP-8433. Analysesusing a DNA sequence input analysis system DNASIS Ver. 3.6 (Takara Bio)revealed a protein of 24.4 kDa with an isoelectric point of 8.50. Theprotein was assumed to be classified into RNase HII based on nucleotidesequence comparisons.

(6) Expression of Archaeoglobus profundus RNase H Gene

Escherichia coli JM109 transformed with pAPR111Nd was inoculated into 10ml of LB medium containing 100 μg/ml of ampicillin and 1 mM IPTG andcultured with shaking at 37° C. overnight. After cultivation, cellscollected by centrifugation were suspended in 176.3 μl of a buffer (20mM tris-HCl (pH 8.0), 1 mM EDTA) and sonicated. A supernatant obtainedby centrifuging the sonicated suspension at 12,000 rpm for 10 minuteswas heated at 70° C. for 10 minutes and then centrifuged again at 12,000rpm for 10 minutes to collect a supernatant as a heated supernatant.Similarly, Escherichia coli HMS174(DE3) transformed with pApr108 wasinoculated into 10 ml of LB medium containing 100 μg/ml of ampicillinand cultured with shaking at 37° C. overnight. After cultivation, cellscollected by centrifugation were processed according to the procedure asdescribed above to obtain a heated supernatant of Apr RNase H.

The enzymatic activities were measured for the heated supernatants asfollows.

1 mg of poly(rA) or poly(dT) (both from Amersham Pharmacia Biotech) wasdissolved in 1 ml of 40 mM tris-HCl (pH 7.7) containing 1 mM EDTA toprepare a poly(rA) solution and a poly(dT) solution.

The poly(rA) solution (to a final concentration of 20 μg/ml) and thepoly(dT) solution (to a final concentration of 30 μg/ml) were then addedto 20 mM HEPES-KOH (pH 7.8) containing 4 mM MgCl₂, 0.1% DMSO and 0.01%BSA. The mixture was reacted at 40° C. for 10 minutes and then cooled to4° C. to prepare a poly(rA)-poly(dT) solution.

1 μl of the heated supernatant of Apr RNase H was added to 100 μl of thepoly(rA)-poly(dT) solution. The mixture was reacted at 40° C. for 10minutes. 10 μl of 0.5 M EDTA was added thereto to terminate thereaction. Absorbance at 260 nm was then measured. As a control, 10 μl of0.5 M EDTA was added to the reaction mixture, the resulting mixture wasreacted at 40° C. for 10 minutes, and the absorbance was then measured.The absorbance for the control was subtracted from the absorbance forthe reaction in the absence of EDTA to determine a value, difference inabsorbance. The concentration of nucleotide released frompoly(rA)-poly(dT) hybrid by the enzymatic reaction was determined on thebasis of the difference in absorbance. One unit of RNase H was definedas an amount of enzyme that increases A₂₆₀ corresponding to release of 1nmol of ribonucleotide in 10 minutes which was calculated according tothe following equation. If a diluted enzyme solution was used, the valueobtained using the equation was corrected based on the dilution rate:Unit=[Difference in Absorbance×Reaction Volume(ml)]/0.0152

As a result, an RNase H activity was observed for the heated supernatantof Apr RNase H.

(7) Preparation of Purified RNase H Preparation

Escherichia coli BL21(DE3) transformed with pApr108 obtained in Example1-(4) was inoculated into 400 ml of LB medium containing 100 μg/ml ofampicillin and cultured with shaking at 37° C. for 17 hours. Aftercultivation, cells collected by centrifugation were suspended in 500 mlof Buffer A [20 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride, 10 mM 2-mercaptoethanol] and sonicated.A supernatant obtained by centrifuging the sonicated suspension at14,000 rpm for 30 minutes was heated at 70° C. for 15 minutes. It wasthen centrifuged again at 14,000 rpm for 30 minutes to collect asupernatant. Thus, 400 ml of a heated supernatant was obtained.

The heated supernatant was subjected to DE52 anion exchange column(Whatman) equilibrated with Buffer A and washed with Buffer A. As aresult, RNase H flowed through the DE52 column.

The protein solution flowed through the DE52 column was subjected toP-II cation exchange column (Whatman) equilibrated with Buffer B (20 mMtris-HCl (pH 7.0), 1 mM EDTA, 100 mM NaCl, 10 mM 2-mercaptoethanol) andeluted with a linear gradient of 100 mM to 1000 mM NaCl. As a result, anRNase H fraction eluted with about 500 mM NaCl was obtained.

150 ml of the RNase H fraction was concentrated to a volume of 50 mlusing polyethyleneglycol (PEG) 20000. 150 mM of Buffer C (20 mM tris-HCl(pH 7.0), 1 mM EDTA, 10 mM 2-mercaptoethanol) was added to theconcentrate, and the mixture was subjected to Heparin-Sepharose OL-6Bheparin affinity column (Amersham BioSciences) equilibrated with BufferB and eluted with a linear gradient of 100 mM to 500 mM NaCl. As aresult, an RNase H fraction eluted with about 250 mM NaCl was obtained.

130 ml of the RNase H fraction was concentrated to a volume of 10 mlusing PEG 20000. The concentrate was subjected to HiLoad 26/60 SuperdexG200HR gel filtration column (Amersham BioSciences) equilibrated withBuffer D (20 mM tris-HCl (pH 7.0), 0.5 mM EDTA, 200 mM NaCl, 10 mM2-mercaptoethanol) and eluted with Buffer D. As a result, 25 ml of anRNase H fraction was obtained.

35 ml of Buffer C was added to 25 ml of the RNase H fraction, and themixture was subjected to SP-Sepharose FF cation exchange column(Amersham BioSciences) equilibrated with Buffer B and eluted with alinear gradient of 100 mM to 500 mM NaCl. As a result, 50 ml of an RNaseH fraction eluted with about 250 mM NaCl was obtained.

50 ml of the RNase H fraction was concentrated to a volume of 11 ml bycentrifugation using Ultrafree-4 BIOMAX-5K (Millipore). 11 ml of theconcentrate was dialyzed against Shape Buffer (25 mM tris-HCl (pH 7.0),0.5 mM EDTA, 30 mM NaCl, 5 mM 2-mercaptoethanol, 50% glycerol), and 4.5ml of an RNase H solution was obtained.

The thus obtained RNase H was used as Apr RNase H preparation.

The enzymatic activity of the Apr RNase H preparation was measured asdescribed in Example 1-(6). As a result, an RNase H activity wasobserved for the Apr RNase H preparation.

Example 2 Homology Search

Homology searches were conducted for the amino acid sequence of RNase Hfrom Archaeoglobus profundus (Apr) obtained in Example 1 and thenucleotide sequence encoding the same. Calculation of homology wasconducted using a computer algorithm FASTA (version 3.2; Pearson, W. R.et al., Pro. Natl. Acad. Sci., 85:2444-2448, 1988) as a search program.

Gene database searches were conducted for Apr RNase H using the computeralgorithm FASTA. As a result, the highest homologies between the aminoacid and nucleotide sequences of Apr RNase H and those of one presumedto be of a ribonuclease were 53% and 60%, respectively.

Example 3 Examination of Thermostability of RNase H

Thermostability of Archaeoglobus profundus RNase H was examined usingEscherichia coli transformed with pApr108 obtained in Example 1-(6). TheE. coli strain was cultured, a crude enzyme extract prepared from theculture was heated at 95° C. for 15 minutes, and the RNase H activitywas determined according to the method as described in Example 1-(6). Asa result, an RNase H activity was observed for the RNase H derived fromArchaeoglobus profundus.

In addition, Apr RNase H at a concentration of 5 units/ml in a heattreatment buffer (25 mM tris-HCl (pH 8.0), 5 mM mercaptoethanol, 30 mMNaCl, 0.5 mM EDTA, 0.1% BSA, 50% glycerol) was heated for 10 minutes at50, 60, 70, 80 or 90° C., and the remaining activity was determined. Thesample with or without heat treatment was added, at a finalconcentration of 0.2 units/ml, to an activity measurement solution (atfinal concentrations, 32 mM HEPES-potassium hydroxide buffer (pH 7.8),100 mM potassium acetate, 1% DMSO, 0.05% BSA, 4 mM magnesium acetate,0.2 μM substrate DNA-RNA-DNA and 1 μM templateW49 (SEQ ID NO:16)). Thedegradation rate per enzyme unit was determined with probeW3 (SEQ IDNO:15). The results are shown FIG. 1, defining the activity of RNase Hwithout heat treatment as 100. Apr RNase H had almost 100% of theactivity after heating at 50 to 80° C., and 86.9±7.0% remaining activityafter reaction at 90° C. for 10 minutes.

Example 4 Examination of HBV Detection Using ICAN System with Apr RNaseH

A 560-bp PCR-amplified fragment corresponding to a part of HBV X proteingene (SEQ ID NO:10) was inserted into pT7Blue T vector by TA cloning.The resulting plasmid was used as an HBV positive control.

The composition of the reaction mixture was as follows: at finalconcentrations, 32 mM HEPES-potassium hydroxide buffer (pH 7.8), 100 mMpotassium acetate, 1% DMSO, 0.01% BSA, 4 mM magnesium acetate, 600 μMeach of dNTPs, 11 units of BcaBEST DNA polymerase, 50 pmol each ofprimers HBVF-2 (SEQ ID NO:11) and HBVR-1 (SEQ ID NO:12), 10³ copies ofthe HBV positive control, and 1.625, 3.25, 6.5 or 13 units of Apr RNaseH (final volume of 25 μl). The reaction mixtures were placed in athermal cycler which had been set at 55° C. and incubated for 60minutes.

After reaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel. As a result, the 76-bpamplification products of interest were observed using the respectiveamounts of RNase H.

The sensitivity for the HBV positive control was examined by carryingout similar experiments using 102 copies, 10 copies or 1 copy of the HBVpositive control. As a result, when 6.5 units of Apr RNase H were added,the highest sensitivity was observed and 1 copy of the HBV positivecontrol could be detected.

Next, RNase HII from Pyrococcus furiosus (Pfu RNase HII), RNase HII fromArchaeoglobus fulgidus (Afu RNase HII) and RNase HII from Thermococcuslitoralis (Tli RNase HII) were prepared as described in WO 02/22831.They were used for detection of HBV using the above-mentioned ICANsystem and the sensitivities were compared with that observed with AprRNase H. When Pfu RNase HII was used, the highest sensitivity wasobserved using 2.2 units of the added enzyme with sensitivity of 10copies of the HBV positive control. When Afu RNase HII was used, thehighest sensitivity was observed using 2.2 units of the added enzymewith sensitivity of 10 copies of the HBV positive control. When TliRNase HII was used, the highest sensitivity was observed using 8 unitsof the added enzyme with sensitivity of 10 copies of the HBV positivecontrol.

As described above, one copy of the HBV positive control could bedetected using Apr RNase H for detection of the HBV gene using the ICANsystem. Thus, it was confirmed that the highest sensitivity could beattained.

Example 5 Comparison of Substrate Degradation Rate in Relation toDifference in Substrate Form for Apr RNase H

Degradation rates of RNase H were determined using one of threeDNA-RNA-DNAs containing different numbers of RNAs in their sequences(probeW1 (SEQ ID NO:13), probeW2 (SEQ ID NO:14) and probeW3 (SEQ IDNO:15)) as a substrate, and the differences were examined. In addition,cleavage rates of RNase HII from Pyrococcus furiosus (Pfu RNase HII) andRNase HII from Pyrococcus horikoshii (Pho RNase HII) prepared asdescribed in WO 02/22831 as well as RNase HI from Thermus thermophilus(Tth RNase HI) (Toyobo) using the above-mentioned substrates werecompared.

If a solution containing probeW1, probeW2 or probeW3 is exposed to lightat wavelength around the maximum excitation wavelength of FAM (495 nm),FAM as a modification at the 5′ end of the substrate emits fluorescenceat the maximum wavelength 519 nm. However, the fluorescence isattenuated as a result of fluorescence resonance energy transfer (FRET)with DABCYL as a modification at the 3′ end. If the substrate is cleavedwith RNase H, the intensity of fluorescence at 519 nm is increased as aresult of relief of FRET. Thus, degradation of the substrate can bemonitored by determining the difference between intensities offluorescence at 519 nm measured before and after cleavage of thesubstrate.

If substrate concentration is sufficiently high as compared with enzymeconcentration, the amount of enzymatic reaction product is increased inproportion to time. If a degradation rate is to be monitored bydetermining fluorescence intensity at 519 nm using a substrate in anamount sufficiently excessive as compared with the amount of enzyme,increase in fluorescence intensity per unit time at the beginning of areaction can be approximated using a linear equation. The slope of theapproximation line corresponds to increase in fluorescence intensity pertime ((fluorescence intensity)/(minute)). Assuming that a substrate in areaction system is completely degraded when increase in fluorescenceintensity reaches a plateau and the fluorescence intensity reaches itsmaximum value, (maximum fluorescence intensity)−(fluorescence intensitybefore cleavage) corresponds to increase in fluorescence intensity peramount of degraded substrate. Reaction rate v ((amount of degradedsubstrate)/(minute)) can be determined based on this value and theincrease in fluorescence intensity per time ((fluorescenceintensity)/(minute)).

Apr RNase H at a final concentration of 0.4, 0.8, 1.6 or 2.4 unit(s)/mlwas added to an activity measurement solution (at final concentrations,32 mM HEPES-potassium hydroxide buffer (pH 7.8), 100 mM potassiumacetate, 1% DMSO, 0.05% BSA, 4 mM magnesium acetate, 0.2 μM substrateDNA-RNA-DNA and 1 μM templateW49 (SEQ ID NO:16)). A real-time PCRmeasurement instrument Smart Cycler (Takara Bio) was used for RNase Hreaction and fluorescence intensity measurement. Reaction rate v((amount of degraded substrate)/(minute)) was determined by conducting areaction at 55° C. for 100 minutes. Reaction rate per enzyme unit((amount of degraded substrate)/(minute·unit)) was determined based on aslope of a calibration curve prepared by plotting reaction rate v((amount of degraded substrate)/(minute)) against Apr RNase Hconcentration. Activity measurements were carried out using Pfu RNaseHII, Pho RNase HII or Tth RNase HI in a similar manner to determinereaction rates per enzyme unit ((amount of degradedsubstrate)/(minute·unit)). In case of Tth RNase HI, Tth RNase HI wasadded to the activity measurement solution at a final concentration of10, 20, 30 or 40 units/ml.

Reaction rates per enzyme unit of the respective enzymes determinedusing probew1, probeW2 or probeW3 are shown in Table 2. Values inpmol/(minute·unit) are shown in the table. Relative reaction ratesdefining reaction rate per enzyme unit using probeW3 as 100% areindicated in parentheses. Using Apr RNase H, cleavage rates higher thanthose observed using other RNase Hs were observed in all cases ofvarious RNA numbers. The highest cleavage rate was observed using thesubstrate having two RNAs. The cleavage rates observed using thesubstrates having one or two RNA(s) were close to the cleavage rateobserved using the substrate having three RNAs.

TABLE 2 probeW1 probeW2 probeW3 (RNA = 1) (RNA = 2) (RNA = 3) Apr RNaseHII 17.0 (61.6) 41.1 (148.9) 27.6 (100)  Pfu RNase HII 3.3 (34.8) 5.3(55.9) 9.5 (100) Pho RNase HII 2.7 (55.6) 3.2 (67.8) 4.8 (100) Tth RNaseHI 0.0 (0) 0.0 (2.2)  1.4 (100)

As described above, it was shown that Apr RNase H whose cleavage rate isless susceptible to influence of RNA length is useful for nucleic acidamplification reactions and nucleic acid detection reactions.

INDUSTRIAL APPLICABILITY

The present invention provides a polypeptide having an RNase H activitywhich is highly valuable for genetic engineering, a gene encoding saidpolypeptide and a method for producing said polypeptide by geneticengineering. Since the RNase H of the present invention is thermostable,the present invention provides a method for producing an RNase H whichis industrially advantageous.

It is now possible to use the RNase H of the present invention forvarious uses according to the present invention.

Sequence Listing Free Text

SEQ ID NO:3: PCR primer RN-F1 for cloning a gene encoding a polypeptidehaving a RNaseH activity from Archaeoglobus profundus

SEQ ID NO:4: PCR primer RN-R2 for cloning a gene encoding a polypeptidehaving a RNaseH activity from Archaeoglobus profundus

SEQ ID NO:5: PCR primer AprRN-1 for cloning a gene encoding apolypeptide having a RNaseH activity from Archaeoglobus profundus

SEQ ID NO:6: PCR primer AprRN-2 for cloning a gene encoding apolypeptide having a RNaseH activity from Archaeoglobus profundus

SEQ ID NO:7: Taq sequence

SEQ ID NO:8: PCR primer AprNde for amplifying a gene encoding apolypeptide having a RNaseH activity from Archaeoglobus profundus

SEQ ID NO:9: PCR primer AprBam for amplifying a gene encoding apolypeptide having a RNaseHII activity from Archaeoglobus profundus

SEQ ID NO:11: Chimeric oligonucleotide primer to amplify a portion ofHepatitis B virus X protein. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:12: Chimeric oligonucleotide primer to amplify a portion ofHepatitis B virus X protein. “nucleotides 20 to 22 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:13: Chimeric oligonucleotide designed as probeW1. “nucleotide9 is ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:14: Chimeric oligonucleotide designed as probeW2. “nucleotides9 to 10 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:15: Chimeric oligonucleotide designed as probeW3. “nucleotides9 to 11 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:16: Oligonucleotide designed as templateW49.

1. An isolated nucleic acid encoding a polypeptide having a thermostableribonuclease H activity, said nucleic acid being selected from the groupconsisting of: (a) a nucleic acid encoding a polypeptide having theamino acid sequence of SEQ ID NO: 1; (b) a nucleic acid encoding apolypeptide having an amino acid sequence that shares at least 85%identity with the amino acid sequence of SEQ ID NO: 1; (c) a nucleicacid having the nucleotide sequence of SEQ ID NO: 2; (d) a nucleic acidcapable of hybridizing to the nucleic acid consisting of the nucleotidesequence of SEQ ID NO:2 or the full-length complementary strand thereofunder conditions of incubation in 6×SSC (1×SSC: 0.15 M NaCl, 0.015 Msodium citrate, pH 7.0) containing 0.5% SDS, 0.1% bovine serum albumin(BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400 and 0.1% denaturedsalmon sperm nucleic acid at 50° C. for 20 hours, and washing in 2×SSCcontaining 0.5% SDS at 37° C. while changing the SSC concentration downto 0.1×SSC and the temperature up to 50° C.; and (e) a nucleic acidhaving a nucleotide sequence that shares at least 90% identity with thenucleotide sequence of SEQ ID NO: 2, wherein said nucleic acid isendogenously found in organisms from the genus Archaeoglobus, which isamplified by PCR using primers having nucleic acid sequences of SEQ IDNO: 8 and SEQ ID NO: 9 and 40 cycles of 94° C. for 30 seconds, 55° C.for 30 seconds and 72° C. for 1 minute.
 2. A recombinant DNA comprisingthe nucleic acid of claim
 1. 3. An isolated transformant transformedwith the recombinant DNA of claim
 2. 4. A method for producing apolypeptide having a thermostable ribonuclease H activity, the methodcomprising: culturing the transformant of claim 3; and collecting apolypeptide having a thermostable ribonuclease H activity from theculture.